Phosphonate analogues of nucleoside polyphosphates

This article provides an overview of the efforts toward the synthesis of nucleoside polyphosphate mimics featuring a P-CXY-P scaffold. The following synthetic approaches to these compounds are summarized: (i) nucleophilic displacement of 5´-O -tosyl nucleoside by the ammonium salts of methylenebisphosphonic acid; (ii) synthesis via activated phosphate/phosphonate substrates; (iii) Mitsunobu coupling between a nucleoside and methylenebisphosphonic acid; (iv) phosphorylation of a protected nucleo side under Yoshikawa’s reaction conditions with methylenebis(phosphonic dichloride); (v) synthesis via nucleophilic cleavage of cyclic trimetaphosph(on)ates; (vi) enzyme-mediated reactions.


Synthesis via 5´-O-tosyl nucleosides
Lipophilic salts of methylenebisphosphonic acid such as tris(tetra-n-butylammonium) bisphosphonate are good reagents for synthesis of nucleotide analogues from nucleosides by direct nucleophilic displacement at the 5´-position of 5´-O-tosyl nucleosides.While this is multi-step synthesis (particularly if protected nucleosides are used), the relative simplicity and high reliability of the procedure make it a good supplement to existing methods based on addition of the nucleophilic 5'-hydroxyl group to activated phosphonate derivatives.
The preparation of methylene-modified nucleoside polyphosphates by the nucleophilic displacement of Osulfonyl groups such as p-toluenesulfonyl (Ts) or methylsulfonyl (Ms), was first recorded by Stock in 1979  when it was reported that the action of trialkylammonium salts of methylenebisphosphonic acids on 5´-O-tosyl thymidine in DMF leads to the corresponding bisphosphonate analogues of thymidine di-and triphosphate 1, 2 (Scheme 1). 25 Later, this approach has been adapted by Poulter and co-workers to the preparation of a variety of nucleoside diphosphates and their bisphosphonate analogues. 26Thus, reactions between 5´-O-tosyl derivatives of adenosine and 2´-deoxyadenosine and tris(tetra-n-butylammonium)salts of methylenebisphosphonic acids in freshly distilled dry acetonitrile afforded the nucleoside bisphosphonates 3-5 in good yields (Scheme 2). 27By essentially the same procedure (sometimes including variations in the reaction conditions), CHF-and CF2-analogues of 2´-deoxythymidine diphosphate 28 , CF2-modified guanosine diphosphate 29 and adenosine 5'-(α,β:β,γ-dimethylenetriphosphate) 30 have been synthesized and characterized by spectral methods.Findings on the selectivity of the series of modified ATPs for rat P2X2 and P2X2/3 receptors are summarized in ref. 15.Scheme 1. Synthesis of bisphosphonate analogues of thymidine di-and triphosphate from 5´-O-tosylthymidine. Scheme 2. Poulter's synthesis of α,β-methylene-modified ADP derivatives.
An attractive feature of Poulter's phosphorylation method is that either protected or unprotected nucleosides can be phosphorylated.Yields are higher for protected nucleosides, suggesting that in appropriate cases it may be worthwhile to use protected strategy in spite of the added synthetic steps. 31Thus, α,βmethylene-modified derivatives of ADP 6-8 were prepared using 2´,3´-O-isopropylidene-adenosine 5´-O-tosylate.After purification of the intermediates they were deprotected by treatment with 6-8% trifluoroacetic acid to obtain desired products (Scheme 3). 32ith a view to set rules for design of UDP-based reversible P2Y6 receptor antagonists as potential drugs, a variety of protected uridine 5´-tosylates were tested in nucleophilic displacement reactions with tetrabutylammonium bisphosphonates giving the desired uracil nucleotide analogues 9-17 after acidic workup (Scheme 4). 33heme 3. Synthesis of α,β-methylene-substituted ADP derivatives via 2´,3´-O-isopropylidene-adenosine 5´-O-tosylate.

Scheme 4. Synthesis of uracil nucleotide analogues.
A new type of nucleoside polyphosphate analogues in which pyrophosphate oxygen is replaced by a potentially reactive carbonyl group was obtained by displacement of the 5´-mesyl group of the corresponding 5´-mesylnucleoside with carbonylbisphosphonate.Reaction of the tributylammonium salt of carbonylbisphosphonate with N 2 -(4-butylphenyl)-5´-mesyl-2´-deoxyguanosine in acetonitrile gave 18 isolated as a yellow solid in 93% by ion-exchange chromatography.This compound was a potent, competitive inhibitor of human DNA polymerase α (Scheme 5). 34Scheme 5. Synthesis carbonylbisphosphonate analogue of BuPdGDP via displacement of 5´-mesyl group.
Blackburn and Langston prepared α,β-substituted phosphonate analogues of 2´-deoxyadenosine and 2´-deoxythymidine 5´-triphosphates by a two-step reaction starting from the appropriate 5´-O-tosyl deoxynucleosides (Scheme 6). 28In the first step, they prepared 2´-deoxynucleoside 5´-diphosphate analogues 19 in yield around 50-60%.The -phosphate group was attached subsequently either via activation of P β of the dNDP as its morpholidate followed by reaction with inorganic phosphate (method A) or phosphorylation of nucleoside 5´-diphosphate with an excess of p-nitrobenzyl phosphoromorpholidate (method B).The p-nitrobenzyl group was removed by catalytic hydrogenolysis to give the dNTP analogues 20 in good yields.Scheme 6. Synthesis of α,β-CXY dNTP analogues by combination of tosylate substitution and phosphoromorpholidate protocol.
α,β:β,γ-BisCF2 substituted RNA nucleotide analogues 21-24 potentially stable to enzymatic hydrolysis in RNA and DNA polymerase assay were prepared via nucleophilic displacement of 5´-tosylate in benzoyl protected nucleosides by the tetra-n-butylammonium salt of bis(difluoromethylene)triphosphonic acid (Scheme 7).Two equivalents of the tosyl nucleoside were required to ensure maximum consumption of triphosphonate salt.In the case of ATP, CTP and UTP nucleotide analogues authors were able to achieve conversion close to 90%, although in case of GTP analogue conversion did not exceed 20%.Preliminary biological results have shown that this class of nucleotides with modified triphosphate moiety revealed the correct polarity and minimal steric effects compared to the natural molecules. 35As part of a program to investigate the mechanism of action of dinucleoside polyphosphate hydrolases, British biochemists described the synthesis of a range of analogues of diadenosine 5´,5´´´-triphosphate (Ap3A). 37The most effective route to compounds 30 involves the condensation of α,β-methylene analogues of ADP, conveniently prepared by the method of Poulter, with adenosine 5´-phosphoromorpholidate (Scheme 9).The α,β:β,-bismethylene analogue 31 was prepared by the reaction between 2´,3´-O-isopropylidene adenosine 5´-tosylate and bis(dihydroxyphosphonomethyl)phosphinic acid.Unfortunately, the yield of pure material was only 3% (Scheme 10). 37Scheme 10.Synthesis of α,β:β,γ-bismethylene Ap3A analogue via 5´-tosylate substitution.
Pankiewicz and co-workers have reported a one-pot reaction involving initial displacement of the mesyl group of 2΄,3΄-O-isopropylidene-5΄-O-mesylthiazofurin (32) with the tris(tetrabutylammonium salt) of difluoromethylenebisphosphonic acid followed by DCC-coupling of compound 33 with nucleoside 34 to give the desired bisphosphonate analogue 35 (Scheme 11).The latter was found to be a potent inducer of differentiation of K562 erythroid leukemia cells. 38heme 11.Synthesis of a nonhydrolyzable CF2-MBP analogue of thiazole-4-carboxamide and benzamide adenine dinucleotide.

Synthesis via activated phosph(on)ate substrates
Diphenyl chlorophosphate [40][41][42][43] , N,N´-carbonyldiimidazole (CDI) 31,44,45 , imidazole/2,2´-dithiopyridine/Ph3P system [46][47][48][49][50] , dicyclohexylcarbodiimide (DCC) 51,52 , and trifluoracetic anhydride 53,54 are the most widely used activating reagents for the synthesis of methylene-modified nucleoside tri-and polyphosphates.All coupling methods have the same strategy in common: one nucleotide subunit (usually nucleoside monophosphate or bisphosphonate) is converted via an activation process into an electrophilic substrate and then reacted with a second phosphate or phosphonate subunit acting as a nucleophile.In principle, two alternative approaches can be used for triphosphate bridge formation.In the first, a nucleotide is activated at the stage of monophosphate and then coupled to a bisphosphonate.In the second, a bisphosphonate is activated and coupled with a nucleoside monophosphate.Thus, the reaction sequences shown in Scheme 14 were the basis of numerous works in which nucleotide 5´-(β,γ-methylene) triphosphates were prepared via activated phosphate/phosphonate substrates. 55heme 14. Simplified general approach for the synthesis of β,γ-methylene nucleoside triphosphates via phosphate/phosphonate activation.
A simple, one-pot method using the diphenyl chlorophosphate technique to prepare β,γ-modified ATP analogues 38 and 39 is illustrated in Scheme 15. 40,56 The reactions proceed at room temperature in Py or Py/DMF solution to give the corresponding products in moderate yields.This approach was the most successful to obtain solid-supported 5´-(α-P-thio) triphosphate oligonucleotide analogues compared to other methods involving the use of phosphoroamidate or salicyl-phosphite intermediates. 41cheme 15.Synthesis of β,γ-CH2-and β,γ-CF2 ATP analogues via (PhO)2P(O)-activated AMP.
Schmitt and Tampé reported an example for the application of diphenyl chlorophosphate activation in a late step of the synthesis of a novel class of nonhydrolyzable ATP-lipids 40 where the nucleotides are covalently attached via C 8 (or N 6 )-position of the adenine ring to a synthetic lipid (Scheme 16). 42Possible applications of the novel class of ATP-lipid have been discussed.
Currently, the most commonly used methods for the preparation of β,-methylene-modified nucleotides involve either a morpholidate or imidazolidate activation.The morpholidate method, introduced by Khorana as one of the first successful strategies for the synthesis of nucleoside-5´-polyphosphates, 58 employs a twostep process which involves conversion of nucleoside monophosphate (NMP) to the corresponding morpholidate via dicyclohexylcarbodiimide (DCC) activation followed by conjugation with the appropriate bisphosphonate.Myers prepared the first bisphosphonate analogue of adenosine 5´-triphosphate, β,-CH2 ATP, by condensing methylenebisphosphonic acid with adenosine 5´-phosphoromorpholidate. 59[62] Scheme 18. Synthesis of β,-methylene bisphosphonate (d)NTP analogues via morpholidate intermediates.
The effective route to analogues of diadenosine 5',5'''-P 1 , P 3 -triphosphate 45 involves the condensation of adenosine C-phosphoromorpholidate 44 with P 1 , P 2 -methylene analogues of ADP 43, conveniently prepared by the method (Scheme 19). 37he search for carbocyclic nucleotides with potent anti-HIV activity led to the synthesis of the pyrophosphoryl phosphonate 48 63,64 and its diphosphonate analogues 49 65,66 with progressive fluorosubstitution within the βγ-methylene linker group as shown in Scheme 20.
Noteworthy features of the chemical syntheses include the transformation of the nucleoside monophosphate 46 into the activated morpholidate 47, and the coupling of 47 with the corresponding bisphosphonate.Nucleotides 50, 51 were found to be potent inhibitors of HIV reverse transcriptase.The three nucleotide triphosphate mimics 52-54 were also synthesized and tested as inhibitors of HIV RT in an enzyme assay.Both 52 and the monofluorinated analogue 53 showed relatively poor activity, being three orders of magnitude less active than the parent compound 48.The difluorinated analogue 54 was markedly more effective than the monofluorinated substrate but was still two hundred times less potent than 48.The disappointing activity of 52-54 may be due to the fact that the carboxy group is a poor mimic of the terminal phosphonate group in compound 51. 66cheme 19.Synthesis of analogues of diadenosine 5',5'''-P 1 , P 3 -triphosphate utilizing the combination of the Poulter method and morpholidate activation.Scheme 20. Preparation of carbocyclic nucleotide analogues with progressive fluoro-substitution within the β,γ-methylene linker group.
Phosphorimidazolidate intermediates result from activation of nucleoside monophosphates with 1,1´carbonyldiimidazole (CDI).Hoard and Ott's original research on this transformation featured syntheses of triphosphates from dNMPs and inorganic pyrophosphate. 68A similar approach is applicable to the synthesis of β,-methylene nucleoside triphosphates.Addition of acidic bisphosphonate activates the phosphorimidazolidate toward nucleophilic displacement giving phosphonate-modified NTP.The imidazolidate method is usually performed as a one-pot synthesis; the nucleoside monophosphate is converted to the imidazolidate by activation with CDI followed by the addition of the appropriate bisphosphonate. 31,44,45omplications in the imidazolidate procedure have been reported when ribonucleosides with unprotected vicinal diols were activated with CDI.This phosgene equivalent easily forms cyclic carbonates that were carried through as impurities in the triphosphorylation procedure.Additionally, nucleoside phosphorimidazolidate can react sluggishly with methylenebisphosphonate, requiring prolonged reaction times or the use of catalysts such as ZnCl2 or CdCl2. 48,69In some cases, reversal of the commonly used strategy, such that the α,βmethylene NDP is the nucleophile and the phosphorimidazolidate is the electrophile, resulting in a high yield of the desired NTP analogue without the need for catalyst or long reaction time.
Ingall et al. reported the synthesis of a series of ATP analogues 64 designed to act as antithrombotic agents.Substitution of the adenine moiety enhanced affinity and selectivity for the P2T receptor and led to the development of a highly potent compound 64a with IC50=0.4 nM.The whole series were prepared via phosphorimidazolidate protocol (Scheme 23). 70ucleotide analogues modified at the glycone and all three phosphate residues were reported by Roberts and co-workers as highly stable in human blood serum with half-lives toward hydrolysis up to 4.5 days. 71These analogues were shown to be selective inhibitors of DNA synthesis, catalyzed by retroviral reverse transcriptases and terminal deoxynucleotidyl transferazes.A typical synthetic procedure is shown for ATP analogues 65 and 66 in Scheme 24.
Scheme 24.Synthesis of glycone and triphosphate modified nucleotide analogues.
The special need for nucleotides with a modified polyphosphate chain as rapid and highly efficient coupling reagents led to the development of an effective method of preparation of phosphorimidazolidate intermediates via the Mukaiyama-Hashimoto oxidation-reduction conditions. 72cheme 25 illustrates the synthetic pathway for obtaining methylene analogue of nucleoside tri-and tetraphosphates.The reaction was performed by activation of a nucleotide unit with imidazole in the presence of triphenylphosphine/2,2´-dithiodipyridine (DTDP) system, followed by coupling with organic salt of bisphosphonate carried out in DMF in a presence of an 8-fold excess of ZnCl2. 697][48][49][50] The coupling reactions occurred efficiently without significant accumulation of by-products which is important because of purification difficulties common for this class of compounds.

Scheme 25. Synthesis of CH2-modified nucleotide analogues via the Mukaiyama-Hashimoto oxidationreduction conditions.
In order to obtain dinucletide cap analogues labeled at the ribose of the 7-methylguanosine moiety with N-methylanthraniloyl, Jemielity and co-workers have used the reverse strategy involving ZnCl2-mediated coupling of bisphosphonate-modified nucleotide P-imidazolidate 67 with fluorescently labeled nucleoside monophosphate 68 (Scheme 26). 73Compound 69 was obtained with a yield of 12% after two purification steps, ion-exchange chromatography and HPLC.
Recently, Sun and co-workers have developed a novel P V -N activation method for the synthesis of nucleoside 5´-triphosphates and their β,-bridging oxygen-modified analogues from nucleoside 5´phosphoropiperidates with 4,5-dicyanoimidazole (DCI) as the activator. 74A high-yielding and chromatographyfree method for preparation of nucleoside 5´-phosphoropiperidates 72-76 is shown in Scheme 28.Scheme 28.Method for synthesis of nucleoside 5'-phosphoropiperidates.
The obtained nucleoside 5´-phosphoropiperidates exhibited excellent reactivity toward bisphosphonate reagents in the presence of 4,5-dicyanoimidazole and afforded β,-CX2-NTP products in high isolated yields (Scheme 29).In the following research, the same authors extended the application of the phosphoropiperidate/DCI system for the preparation of symmetrical and asymmetrical P 2 ,P 3 -CX2-dinucleoside tetraphosphates (Scheme 30).Compared to the conventional phosphoromorpholidate method, this approach afforded products in shorter reaction time and higher isolated yields. 75A one-pot method for DCI-promoted synthesis of symmetrical NppCX 2 ppN bisphosphonate analogues simply from nucleoside 5´-phosphoropiperidates without using nucleoside phosphonates has also been described. 76 very effective synthetic procedure for the preparation of a series of dinucleoside tetraphosphate analogues via activated bisphosphonates and nucleoside monophosphates was developed by Yanachkov et al. 77 They found that organic salts of pyrophosphoric acid and its halomethylenebisphosphonate analogues react with an excess of 1,1‫-׳‬carbonyldiimidazole (CDI) to give stable, isolable diimidazolidates 77, and that these diimidazolidates react with nucleoside 5´-monophosphates or monothiophosphate 78 to give the corresponding nucleotide analogues 79 conveniently and in high yield (Scheme 31).Several bisphosphonate analogues of P 1 ,P 4 -di(adenosine-5´) tetraphosphate were evaluated with respect to their effects on platelet aggregation and function of the platelet P2Y1, P2Y12, and P2X1 receptors.Some of the compounds showed very potent (nanomolecular level) inhibition of ADP induced human platelet aggregation, thus presenting a new and promising class of antiplatelet drugs (Figure 1). 78 The activation strategy in which a bisphosphonate is activated with imidazole and then coupled with a non-activated nucleotide has been applied by Polish researchers to the synthesis of di-(7-methylguanosine)-tetraphosphates and their α,δ-diborano and α,δ-dithio analogues 80, 81 containing a β,γ-methylene group (Scheme 32). 79heme 32.Synthesis of dinucleotide cap analogues modified within a polyphosphate chain.

Synthesis via the Mitsunobu reaction
Discovered in 1967 by Oyo Mitsunobu, this mild multicomponent reaction permits esterification of an acidic component (HX, pKa < 11) with a primary or a secondary alcohol (ROH) in the presence of triphenylphosphine and diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD) (Scheme 33). 80,81heme 33.The Mitsunobu reaction Coupling of nucleosides with phosphoric or phosphonic acids using the Mitsunobu reaction, pioneered by Mioskowski and co-workers, 82 has become one of the important methods available for the synthesis of nucleoside polyphosphates and polyphosphonates.The earliest report of the effectiveness of the Mitsunobu condensation in nucleoside phosphonate chemistry was the preparation of 6-chloroadenosine α,β:β,bismethylenetriphosphate analogue 82 (Scheme 34). 83The original procedure involves treatment of phosphonic acid salt (1 equiv), nucleoside (1 equiv), and triphenylphosphine (3 equiv) in anhydrous pyridine with HBF4 (1 equiv) followed by dropwise addition of DEAD (3 equiv).Scheme 34.Synthesis of a 6-chloroadenosine α,β:β,-bismethylenetriphosphate analogue.
The same strategy, namely the Mitsunobu condensation of tribenzyl methylenebisphosphonate with protected guanosine 83, has been utilized for the synthesis of nucleoside bisphosphonate 85.Hydrogenolysis of compound 84 was achieved using a mixture of Pd/C and Pearlman catalyst 84 (Scheme 35).

Scheme 35. Synthesis of 9-[5´-O-(methylenebisphosphonate)-β-D-ribofuranosyl]guanine.
An attractive feature of Mitsunobu's phosphorylation is the good or high yield of products for some nucleoside substrates.Disadvantages of the method are a lack of tolerance to purine bases and the difficulties that may be encountered in the synthesis of the suitable methylene phosphate analogues.3][84][85] Another possible side reaction involves formation of the 5´-hydrazo substituted compounds, which are most likely formed due to the rearrangement of an intermediate nucleoside-PPh3/DEAD complex. 85Moreover, the Mitsunobu reaction is a poor choice if an incoming phosphonate substitute has several hydroxyl groups available for coupling. 86,87Nevertheless, in spite of these limitations, several recent innovations have significantly extended the scope and synthetic utility of the method. 86,88,89Thus, Taylor and co-workers have developed an unsymmetrical approach to the synthesis of bismethylene triphosphate analogue 86 via sequential Michaelis-Arbuzov reactions on bis-halomethylenephosphinates.The ester 86 was monodeprotected at one of the terminal phosphonate groups by reaction with 1.0 equiv of KCN in DMF at 70 o C. The resulting monodeprotected compounds 87 were used to achieve the first synthesis of the bismethylene analogues of UTP and CTP.Acid 87a was coupled to 2´,3´-O,N 3 -tribenzoyluridine 88 via the Mitsunobu reaction to give 90.However, while this reaction smoothly proceeded to give 79% yield of the product, the authors had to use the triethylammonium salt 87b to get a good yield in the case of 2´,3´-O,N 3 -tribenzoylcytidine 89.Complete deprotection of 90 and 91 was achieved by subjecting them to bromotrimethylsilane followed by treatment with aq.NH4OH-MeOH (Scheme 36). 86heme 36.Synthesis of the bismethylene analogues of UTP and CTP.
Another example of successful application of the Mitsunobu coupling is the synthesis of bismethylene triphosphate nucleotides of uridine 4-phosphate analogues 101.Bismethylene triphosphate derivative 97, a phosphorus component in the synthesis of 101, was prepared by a Michaelis-Arbuzov route from compound 94.The selective cleavage of 5´ ester moiety of 2´,3´,5´-tri-O-acetyl or tri-O-benzoyl U-4-P analogues 98 was accomplished with the aid of a tin catalyst.The Mitsunobu coupling of 5´-deprotected U-4-P analogues 99 to an unsymmetrical bismethylene triphosphate bearing a free phosphonic acid moiety at one of the terminal positions gave fully protected bismethylene triphosphate U-4-P analogues 100.Global deprotection of nucleotides 100 was carried out by treatment with 6-9 equiv of TMSBr followed by ammonium hydroxide in methanol (Scheme 37). 87Scheme 37. Synthesis of bismethylene triphosphate nucleotides of uridine 4-phosphate analogues.
To prepare enzymatically and chemically non-hydrolyzable analogues of dinucleoside triphosphates Ap3A and Gp3G, Lebeau and co-workers have developed a new methodology based on O,O-dialkyl selenophosphonate chemistry. 85,90The bisphosphonic acid 102, a key building block in the synthesis of dinucleoside triphosphate analogues ApCH2pCH2pA and GpCH2pCH2pG, was prepared via a one-pot condensation / transesterification / oxidation / dealkylation sequence involving O,O-dialkyl methaneselenophosphonates. The bisphosphonic acid was then condensed with 2´,3´-O-benzylidene-6-chloroadenosine 103 under modified conditions of the Mitsunobu reaction to afford dinucleoside triphosphate analogue 104 in 40% yield (Scheme 38).The diguanosine derivative was prepared using a similar strategy.
The Mitsunobu esterification was found to be particularly effective for the preparation of potential bisubstrate inhibitors of Leishmania elongating α-D-mannosyl phosphate transferase. 91Thus, coupling between the phosphonodisaccharide methylenebisphosphonate derivative 105 and the guanosine derivative 106 is a crucial step of the synthesis of the required transition state analogue 107 in which a guanosine moiety is linked to the acceptor substrate through the methylenebisphosphonate bridge, mimicking the important guanosine-pyrophosphate motif present in the natural substrate donor GDP-mannose (Scheme 39).

Electrophilic phosphorylation of nucleosides by the Yoshikawa and Ludwig-Eckstein approaches
The Yoshikawa procedure involves the selective 5´-monophosphorylation of a nucleoside with the electrophilic phosphorus oxychloride (POCl3) using trimethyl or triethyl phosphate as the solvent (Scheme 40). 92© ARKAT USA, Inc Yoshikawa's initial studies were performed on 2´,3´-O-isopropylidene-protected NTPs, but later it was found that selective reaction at the 5´-OH was possible for unprotected NTPs and dNTPs.An acidic medium was reported to be critical for selective reaction at the 5´-hydroxyl for unprotected NTPs and dNTPs.In particular, the addition of water to the phosphorylating reagent results in selective 5´-phosphorylation of nucleosides in moderate to high yield. 93Nevertheless, the literature data on the phosphorylation with POCl3 are contradictory and reveal that good regioselectivity can be also obtained when the medium is slightly basic, so the relationship between regioselectivity of phosphorylation and pH remains unclear. 55Yoshikawa and coworkers also used pyrophosphoryl chloride in place of POCl3 but reported no significant advantages. 93hiophosphoryl derivatives of nucleotides can be also obtained via a Yoshikawa procedure that employs PSCl3 to generate 1-thiotriphosphates. 94 Scheme 40.Yoshikawa approach for the synthesis of nucleoside monophosphates.
An important feature of Yoshikawa monophosphorylation reactions is that phosphorodichloridate intermediates can be used directly for the synthesis of nucleoside triphosphates.Ludwig 103 and Ruth 94 have shown that treatment of phosphorochloridates, generated in situ via Yoshikawa procedure, with bis(tri-nbutylammonium) pyrophosphate in dry DMF affords the nucleoside triphosphates in good yields.This approach was successfully adopted for the synthesis of β,-methylene-substituted nucleoside triphosphates.Thus, Fisher and co-workers have proposed a short one-pot synthesis of 2-MeS-β,-CH2-ATP (116) represented in Scheme 45. 104 To ensure a selective reaction of 2-methylthioadenosine at 5´-OH, they used 2´,3´methoxymethylidene-2-methylthioadenosine 114 as the starting material.Nucleoside 114 was first treated with Cl3PO in (MeO)3PO in the presence of 1,8-bis(dimethylamino)naphthalene (proton sponge), followed by the addition of bis(tributylammonium)methylenebisphosphonate and tributylamine.Finally, hydrolysis of the cyclic intermediate 115 and deprotection of the methoxymethylidene group afforded 116 in 35% overall yield.Scheme 45.Application of Yoshikawa approach to the synthesis of a β,-CH2 ATP analogue.
The fact that P2Y receptors have been found to be implicated in a variety of pathophysiological states such as vascular, inflammatory, and immune diseases pushed Müller's team to synthesize a series of UTP, UDP, and UMP derivatives and analogues modified in the uracil part of the molecule. 105Thus, a triphosphate-analogous structure containing a β,-dichloromethylene bridge was successfully introduced into 5-bromouridine via Yoshikawa approach, yielding the nucleotide analogue 117 as shown in Scheme 46.A β,-dichloromethylene modification in the triphosphate chain of 5-bromo-UTP was tolerated by all three receptor subtypes, thus opening up a new strategy to obtain ectonucleotide diphosphohydrolase-and phosphatase-resistant P2Y2, P2Y4, and P2Y6 receptor agonist.Scheme 46.Synthesis of β,-dichloromethylene-substituted 5-bromo-UTP analogue.
A similar procedure was used by Müller and co-workers for the preparation of new β,-CCl2 substituted ATP based 3 H-labeled radioligand 120 ([ 3 H]PSB-0413) (Scheme 47). 106As a precursor for tritiation authors selected the corresponding propargyl derivative.Reaction of 118 with phosphorus oxychloride in trimethyl phosphate followed by reaction with dichloromethylenediphosphonic acid in DMF afforded the corresponding triphosphate analogues 119.The latter was subsequently subjected to catalytic hydrogenation using tritium gas.In preliminary saturation binding studies, [ 3 H]PSB-0413 showed high affinity for platelet P2Y12 receptors with a KD value of 4.57 nM.Scheme 47. Synthesis of nucleotide analogue [ 3 H]PSB-0413 via the Yoshikawa procedure.
In 1989, Ludwig and Eckstein published a modification of electrophilic phosphorylation that employs 2chloro-4H-1,3,2-benzodioxaphosphorin-4-one. 107This reaction gave an activated phosphite that was reacted © ARKAT USA, Inc with pyrophosphate to form the cyclic intermediate.The latter can be oxidized and hydrolyzed to give the corresponding triphosphate (Scheme 48).It was shown that protection of nucleobase functionality for A, T, G, and C was not required, but selectivity for the 5´-hydroxyl in the initial phosphitylation step was marginal if the 3´-and the 2´-hydroxyl were not protected. 55heme 48.Ludwig-Eckstein electrophilic phosphorylation of nucleosides.Scheme 49.Synthesis of AZT 5´-α-P-borano-β,-bridge-modified triphosphates.
The usefulness of the Ludwig-Eckstein approach in the development of a convenient synthetic route to β,-methylene modified nucleotides was demonstrated by Wang and co-workers who reported the synthesis of AZT 5´-triphosphate mimics 123. 108Thus, reaction of AZT with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4one, followed by treatment of the phosphite intermediate 121 with a bisphosphonate salt, yielded the cyclic triphosphate analogues 122, which were subjected to boronation and subsequent hydrolysis to give AZT 5´-α-P-borano-β,-bridge-modified triphosphates 123 in moderate to good yields (Scheme 49).
Synthesis of a series of 2΄,3΄-dideoxynucleoside 5΄-α-P-borano-β,γ-(difluoromethylene)-triphosphates, ddN5΄-αB-β,γ-CF2 TPs, and their inhibitory properties on HIV-1 RT have also been studied (Scheme 51). 109ompounds 126 were prepared according to a similar procedure for preparation of AZT 5΄-αB-β,γ-CF2 TPs (see Scheme 49).However this synthetic route did not apply well to the nucleosides having an exocyclic amino group; therefore, an alternative synthetic procedure was developed (Scheme 52).The course of the reactions was similar to that in Scheme 49 except that the bis(diisopropylamino)phosphites were the active phosphite intermediates.Treatment of 127 with bis(tributylammonium) difluoromethylenediphosphonate presumably yielded the cyclic intermediates 128, which were subsequently subjected to boronation and hydrolysis to give 129.All the resulting ddN5΄-αB-β,γ-CF2 TPs demonstrated essentially the same level of inhibition of HIV-1 RT as the corresponding ddNTPs.Given their enhanced biological stability, these compounds represent a new class of potential antiviral agents. 109cheme 51.Synthesis of the triphosphate mimics of antiviral 2',3'-dideoxynucleosides.Scheme 52.Synthetic pathway for the preparation of ddN 5′-αB-β,γCF2 TPs.
The modified Ludwig-Eckstein protocol with methylenebisphosphonic acids was also successfully employed for the preparation of AZT tetraphosphate mimics 132 as depicted in Scheme 53.Oxidation of phosphite intermediates with sulfur followed by condensation of 130 with H-phosphonate monoesters 131 (in the presence of excess S8) opens a route to nucleotide analogues AZTpSpCX2ppSA containing two outer thiophosphate moieties and a central bisphosphonate, and related compounds AZTpSpCX2ppSAZT with AZT at both ends.This family of compounds is a hydrolysis-resistant version of the AZTppppA that results from excision of AZT by AZT-resistant HIV reverse transcriptase and therefore may be useful in drug design. 110cheme 53.Synthesis of AZT tetraphosphate mimics via the modified Ludwig-Eckstein procedure.

Synthesis involving nucleophilic cleavage of cyclic trimetaphosph(on)ates
Kenyon et al. reported the synthesis of the anhydride of bismethylenetriphosphonic acid 133 via DCCmediated condensation of bismethylenetriphosphonic acid, HO(O)P[CH2P(O)(OH)2]2. 111The same group has described ring-opening of 133 by 2´,3´-isopropylideneadenosine in a polar aprotic solvent at an elevated temperature in the presence of a strong acid.This led to a simple synthesis of α,β;β,-bismethylene analogue of ATP 134 as shown in Scheme 54. 112 Attempts to use this approach for the synthesis of phosphonate analogue of thymidine triphosphate were unsuccessful. 25cheme 54.Synthesis of α,β;β,-bis-CH2 ATP via anhydride of bismethylenetriphosphonic acid.
Another approach to methylene-modified nucleoside polyphosphates via ring-opening reactions is based on the reactions of a P 1 ,P 3 -cyclic nucleoside trimetaphosph(on)ates which can be prepared by treatment of the corresponding nucleoside triphosph(on)ate analogues with carbodiimides or phosphitylation of the 5´hydroxy group of 2´,3´-protected nucleosides, followed by double substitution of salicylate with bisphosphonates and oxidation of the resulting cyclic phosphite (see also Section iv).For example, an effective method for the synthesis of bis-nucleoside tetraphosphate analogues 135 involves the treatment of nucleoside trimetaphosph(on)ates with nucleoside monophosphates or monothiophosphates (Scheme 55). 77heme 55.Synthesis of bis-nucleoside tetraphosphate analogues from nucleoside P 1 ,P 3 -cyclic triphosph(on)ates.Scheme 56.Synthesis of methylenebisphosphonate analogues of P 1 ,P 2 -disubstituted pyrophosphates via bicyclic trisanhydrides.
Pankiewicz and co-workers have developed a synthesis of the novel nucleoside bicyclic trisanhydrides 136 in the reaction of nucleoside-5´-methylenebisphosphonates with DCC.These authors took advantage of generated anhydrides as intermediates in the synthesis of methylenebisphosphonate analogues of P 1 ,P 2disubstituted pyrophosphates.Thus, the reaction of 136 with benzyl 2´,3´-O-isopropylidene-β-D-ribose followed by hydrolysis and deprotection afforded ADP-ribose analogue 137 in 72% overall yield.Treatment of 136 (R = C Ac ) with N-acetylethanolamine or 1,2-dipalmitoyl-sn-glycerol gave methylenebisphosphonate analogues of CDP-ethanolamine and CDP-DAG (138 and 139, respectively), in high yield (Scheme 56). 113

Enzyme-mediated reactions
Several review articles highlight developments in this field. 17,55,114Enzymatic phosphorylation was shown to be an ideal method for certain applications.However, enzyme-mediated reactions do not allow their routine use for the synthesis of nucleotides with unnatural base, sugar and polyphosphate residues.Worth quoting are Burgess and Cook who noted that "enzyme-mediated syntheses of unnatural nucleoside triphosphates are only cost-effective if the expected advantages of this approach are likely to offset the costs of the additional development time required". 55The merits of enzymatic methods are their minimal need for protection / deprotection steps and the regio-and stereo-chemical unambiguity of biocatalytic reactions.A combination of chemical and enzymatic methods has particular utility in cases where the reactivity of the nucleobase precludes the use of electrophilic phosphorylation reagents.An elegant example of this technique was reported by Slama and co-workers who carried out the synthesis of the two novel cyclic ADP-ribose (cADPR) analogues 142 and 143 via cyclization of the corresponding linear nicotinamide adenine dinucleotides (NADs) 140 and 141 catalyzed by Aplysia californica ADP-ribosyl cyclase (Scheme 57). 102heme 57.Aplysia ADP-ribosyl cyclase-catalyzed synthesis of cADPR[CH2] (142) and 3-deaza-cADPR[CH2] (143) from their linear precursors.
Prior to this study it had been shown that cyclic ADP-ribose (cADPR) is a natural metabolite of NAD and a potent calcium-releasing second-messenger. 115Substitution of the bridging pyrophosphate oxygen with methylene group resulted in compounds that are full agonist but with decreased agonist potency.These nucleotide analogues can be useful as a starting point for the development of membrane permeant cADPR prodrugs. 102arious kinases have been used in biocatalytic conversions of nucleoside diphosphates to the corresponding triphosphates. 30,51,116,117In the example depicted in Scheme 58, synthesis of α,β-methylene-2´deoxynucleoside 5´-triphosphates involves preparation of 2´-deoxynucleoside 5´-diphosphate precursors followed by an enzymatic -phosphorylation. 118Enzymatic phosphorylation has been shown to be more efficient than the chemical approach for preparation of α,β-methylene-dNTPs.The α,β-methylene dNDP analogues examined are poor substrate for pyruvate kinase (PK).Therefore, the authors employed the substrate nonspecific nucleoside diphosphate kinase (NDPK).All synthesized α,β-methylene-dNTPs were found to be potent inhibitors of polymerase β, with Ki values ranging 1-5 μM.McKenna and co-workers have recently described the synthesis of α,β-difluoromethylene deoxynucleoside 5´-triphosphates (α,β-CF2 dNTPs, N = A or C) using a modified chemical-enzymological approach. 16They first converted dA or N 4 -benzoyl-dC to the corresponding 5´-tosylates 144 or 147, respectively, by reaction with tosyl chloride in pyridine.The tosylates were converted to the dNTP α,β-CF2 analogues 145 or 148 via condensation with the tris(tetrabutylammonium) salt of difluoromethylenebisphosphonic acid.Phosphorylation to the dNTP analogues 146 or 149 was achieved using nucleoside diphosphate kinase and a catalytic amount of ATP, regenerated with 2.5 eq. of phosphoenolpyruvate (PEP) with pyruvate kinase (PK) in 50 mM HEPES buffer (Scheme 59).The latter modification renders unnecessary the use of an affinity column to purify the product from excess ATP.

Conclusion
In 2016, gem-bisphosphonates celebrated 45 years of application in medicinal chemistry, but in view of the burgeoning interest in bisphosphonate drugs and the concomitant desirability of expanding the structural scope of this class, it is not surprising that the synthesis of nucleoside polyphosphate analogues containing the P-CXY-P structural motif remains a challenging topic, and the development of highly efficient methodologies for synthesis of these species is of significant importance in biology and medicine.Landmarks in the development of contemporary chemistry of bisphosphonate analogues of nucleoside polyphosphates include Blackburn's synthesis of β,γ-fluoromethylene-bridged analogues of adenosine triphosphate and guanosine triphosphate (1984), Wang's synthesis of 2´,3´-dideoxynucleoside 5´-α-P-borano-β,γ-(difluoromethylene)triphosphates (2005), Prakash's synthesis of fluorinated deoxynucleoside analogues based on bis(difluoromethylene)triphosphoric acid (2010), Pankiewicz's synthesis of the mycophenolic adenine dinucleotide as a potent inhibitor of hIMPDH and leukemia K562 cells proliferation (2011), and McKenna's and Goodman's synthesis of the first individual β,-CHX-dGTP diastereomers [(R)-or (S)-CHX, where X is F or Cl] and determination their structures in ternary complexes with DNA polymerase β (2012).An area of great promise remains regio-and diastereoselective synthesis of nucleoside polyphosphate analogues with CXY-modified phosphate chains; much attention will be focused in the recent years to realize this problem.Moreover, in the next few years further studies to obtain details concerning the interactions of such species with enzymatic binding partners are likely to be the next challenge in bioorganic chemistry.

Figure 1 .
Figure 1.Structures of bisphosphonate Ap 4 A analogues presenting a new promising class of antiplatelet drugs.

Scheme 41 .
Scheme 41.Electrophilic phosphorylation of nucleosides by the Yoshikawa approach.

Valery
Kukhar was born in Kiev, Ukraine, in 1942.He graduated from Dnepropetrovsk Institute of Chemical Technology in 1963, and received his Cand.Chem.Sci.degree in 1967 under the supervision of Professor Alexander Kirsanov from Institute of Organic Chemistry of National Academy of Sciences of Ukraine.He received his Doctor of Chemistry degree in 1974 from Institute of Organic chemistry.In 1978-1988 he was the Chief of Chemical Department of National Academy of Sciences of Ukraine (NASU).Since 1987, he has been Director of Institute of Bioorganic Chemistry and Petrochemistry of NASU.Professor Valery Kukhar is a member of National Academy of Sciences (1985) and he was President of Ukrainian Chemical Society from 1992 to 2002.His research interests concentrated mainly on organophosphorus and organofluorine chemistry.He is the author and editor of 6 books, including Chemistry of Fluorine-Containing Amino Acids (1994) and Aminophosphonic and Aminophosphinic Acids.Chemistry and Biological Activity (2000).He was recipient of GLOBAL -500 Prize (UNEP, 1993), San-Valentino Award (World Federation of Scientists, 1999), and Ukrainian State Award in Science & Technology (1999).Valery Kukhar is a member of OPCW Scientific Advisory Board and International Advisory Group for Chernobyl Shelter Fund, EBRD.